Parabacteroides massiliensis sp. nov., a new bacterium isolated from a fresh human stool specimen

Parabacteroides massiliensis sp. nov., strain Marseille-P2231T (= CSURP2231 = DSM 101860) is a new species within the family Tannerellaceae. It was isolated from a stool specimen of a 25-year-old healthy woman. Its genome was 5 013 798 bp long with a 45.7 mol% G+C content. The closest species based on 16S rRNA sequence was Parabacteroides merdae strain JCM 9497T with 98.19% sequence similarity. Considering phenotypic features and comparative genome studies, we proposed the strain Marseille-P2231T as the type strain of Parabacteroides massiliensis sp. nov., a new species within the genus Parabacteroides.


Introduction
Currently, the genus Parabacteroides includes eight valid species with standing in nomenclature [1]. Among them, Parabacteroides distasonis, Parabacteroides goldsteinii and Parabacteroides merdae previously belonged to the genus Bacteroides but were reclassified as members of the genus Parabacteroides since 2006 [2]. The species Parabacteroides faecis [3] and Parabacteroides johnsonii [4] (faeces) and Parabacteroides gordonii (blood) [5] were all isolated for the first time in humans. Culturomics is a concept developing different culture conditions to enlarge our knowledge of the human microbiota through the discovery of previously uncultured bacteria [6][7][8][9]. Once it was isolated, we used a taxono-genomics approach including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), phylogenetic analysis, main phenotypic description and genome sequencing, to describe this strain [10,11]. Here we describe a new Parabacteroides massiliensis sp. nov., strain Marseille-P2231 T (= CSURP2231 = DSM 101860) according the concept of taxono-genomics.

Isolation and growth conditions
In 2017, we isolated from a fresh stool sample of a 25year-old healthy woman an unidentified bacterial strain. Screening was performed using MALDI-TOF MS on a Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany) as previously described [12]. The obtained spectra ( Fig. 1) were imported into MALDI BIOTYPER 3.0 software (Bruker Daltonics) and analysed against the main spectra of the bacteria included in two databases (Bruker and the constantly updated MEPHI databases). The study was validated by the ethics committee of the IHU Méditerranée Infection under number 2016-010. Initial growth was obtained after 72 hours of culture in a Colombia agar enriched with 5% sheep's blood (bioMérieux, Marcy l'Etoile, France) in strict anaerobic conditions at 37°C and pH 7.5.

Strain identification
The 16S rRNA gene was sequenced to classify this bacterium. Amplification was carried out using the primer pair fD1 and rP2 (Eurogentec, Angers, France) and sequencing using the Big Dye® Terminator v1.1 Cycle Sequencing Kit and ABI Prism 3130xl Genetic Analyzer capillary3500xLGenetic Analyzer capillary sequencer (Thermofisher, Saint-Aubin, France), as previously described [13]. The 16S rRNA nucleotide sequences   were assembled and corrected using CODONCODE ALIGNER software (http://www.codoncode.com). Strain Marseille-P2231 T exhibited a 98.19% sequence identity with Parabacteroides merdae strain JCM 9497 T (GenBank accession number NR_041343), the phylogenetically closest species with standing in nomenclature (Fig. 2). We consequently classify this strain as a member of a new species within the family Tannerellaceae, phylum Bacteroidetes.

Phenotypic characteristics
Colonies were circular and smooth with a mean diameter of 1.2 mm. Bacterial cells were Gram-negative, rod-shaped, ranging in length from 1.27 to 2.46 μm and in width from 0.45 to 0.73 μm (Fig. 3). Strain Marseille-P2231 T showed catalase-negative and oxidase-negative activities. Main phenotypic properties of strain Marseille-P2231 T were studied by using the API 50 CH strips (Table 1), API ZYM strips ( Table 2) and API 20A strips ( Table 3). The main characteristics of strain Marseille-P2231 T are summarized on digitalized protologue (www.imedea.uib.es/ dprotologue) under the number TA00985. The biochemical and phenotypic features of strain Marseille-P2231 T were compared with those of other close representative strains in the Porphyromonadaceae family (Table 4) Cellular fatty acid methyl ester analysis was performed by gas chromatography/mass spectrometry. Two samples were prepared with approximately 5 mg of bacterial biomass per tube harvested from several culture plates. Fatty acid methyl esters were prepared as described by Sasser [14]. Gas chromatography/mass spectrometry analyses were performed as described elsewhere [15]. The most abundant fatty acid by far was 12-methyl-tetradecanoic acid (43%), followed by 3-hydroxy15-methyl-hexadecanoic acid (19%) and hexadecanoic acid (10%). Several branched structures and specific 3-hydroxy fatty acids were described. Minor amounts of unsaturated and other saturated fatty acids were also detected ( Table 5).

Genome sequencing
Genomic DNA was extracted using the EZ1 biorobot (Qiagen, Courtaboeuf, France) with the EZ1 DNA tissue kit and then sequenced using MiSeq technology (Illumina, San Diego, CA, USA) with the Nextera Mate Pair sample prep kit (Illumina), as previously described [16]. The assembly was performed with a pipeline incorporating different software (VELVET [17], SPADES [18] and SOAP DENOVO [19]), and trimmed data (MISEQ and TRIMMOMATIC [20] software) or untrimmed data (only MISEQ software). GAPCLOSER was used to reduce assembly gaps. Scaffolds <800bp in length and scaffolds with a depth value <25% of the mean depth were removed. The best assembly was selected using different criteria (number of scaffolds, N50, number of N). The genome of strain Marseille-P2231 T is 5 013 798 bp long (23 scaffolds, 27 contigs, 762 401 N50) with a 45.7 mol% G+C content and contains 4 195 predicted genes. The degree of genomic similarity of Marseille-P2231 T with closely related species was estimated using the ORTHOANI software [21]. Values among closely related species (Fig. 4) ranged from 70.20% between Parabacteroides massiliensis and Parabacteroides chartae to 91.01% between P. merdae and P. johnsonii. When the isolate was compared with these closely related species, values ranged from 70.20% with P. chartae to 88.73% with P. merdae.   Cell diameter (μm) 0.4-0.7 0.8-1.6 0.8 0.8  Nucleotide sequence accession number The 16S rRNA gene and genome sequences were deposited in GenBank under accession number LN899828, and FTLH00000000, respectively.

Deposit in culture collections
Strain Marseille-P2231 T or strain SN4 T was deposited in strain collection under number (= CSURP2231 T = DSM 101860).
the Hitachi Corporation for providing the TM4000Plus Tabletop microscope. They also thank Aurelia Caputo for submitting the genomic sequences to GenBank.